Synthesis of fluorescent lactosylceramide stereoisomers

Article abstract of DOI:10.1016/j.chemphyslip.2006.03.001

The intracellular distribution of synthetic glycosphingolipids (GSLs) bearing a fluorophore can be monitored in living cells by fluorescence microscopy. We reported previously that variation in the length of the long-chain base and in the structure of the carbohydrate-containing polar head group of (2S,3R) (or d-erythro-)-β-lactosylceramide (LacCer) did not alter the mechanism of endocytic uptake from the plasma membrane of various mammalian cell types [Singh, R.D., Puri, V., Valiyaveettil, J.T., Marks, D.L., Bittman, R., Pagano, R.E., 2003. Selective caveolin-1-dependent endocytosis of glycosphingolipids. Mol. Biol. Cell 14, 3254-3265]. To extend our examination of the molecular features in LacCer that are responsible for its uptake by the caveolar-requiring endocytic pathway, we have synthesized the three unnatural stereoisomers [(2R,3R)-, (2S,3S)-, and (2R,3S)] of dipyrromethene difluoride (BODIPY)-LacCer. These analogues will be used to probe the role of stereochemistry in the long-chain base of LacCer in the mechanism of endocytic uptake.

Full text of DOI:10.1016/j.chemphyslip.2006.03.001

Chemistry and Physics of Lipids 142 (2006) 58–69
Synthesis of fluorescent lactosylceramide stereoisomers
Yidong Liu, Robert Bittman∗
Department of Chemistry and Biochemistry, Queens College of the City University of New York, Flushing, NY 11367-1597, USA
Received 9 August 2005; received in revised form 2 March 2006; accepted 3 March 2006
Available online 29 March 2006
Abstract
The intracellular distribution of synthetic glycosphingolipids (GSLs) bearing a fluorophore can be monitored in living cells by
fluorescence microscopy. We reported previously that variation in the length of the long-chain base and in the structure of the
carbohydrate-containing polar head group of (2S,3R) (or d-erythro-)-␤-lactosylceramide (LacCer) did not alter the mechanism of
endocytic uptake from the plasma membrane of various mammalian cell types [Singh, R.D., Puri, V., Valiyaveettil, J.T., Marks, D.L.,
Bittman, R., Pagano, R.E., 2003. Selective caveolin-1-dependent endocytosis of glycosphingolipids. Mol. Biol. Cell 14, 3254–3265].
To extend our examination of the molecular features in LacCer that are responsible for its uptake by the caveolar-requiring endocytic
pathway, we have synthesized the three unnatural stereoisomers [(2R,3R)-, (2S,3S)-, and (2R,3S)] of dipyrromethene difluoride
(BODIPYTM)-LacCer. These analogues will be used to probe the role of stereochemistry in the long-chain base of LacCer in the
mechanism of endocytic uptake.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Fluorescent lipid analogues; Glycosphingolipids; Lipid synthesis
1. Introduction
MalCer, utilized the same caveolar-dependent endo-
cytic pathway for uptake from the plasma membrane
of different cells (Singh et al., 2003; Bittman, 2004).
In contrast, BODIPYTM-sphingomyelin utilizes both a
clathrin-dependent and a caveolar-dependent pathway in
approximately equal extents for internalization (Puri et
al., 2001). To examine the role of stereochemistry at C2
and C3 of the sphingosine chain of LacCer in determin-
ing the mechanism of endocytosis, we have prepared the
following unnatural stereoisomeric analogues: (2R,3R)-,
(2S,3S)-, and (2R,3S)-BODIPYTM-LacCer (compounds
b–d in Fig. 1).
A boron dipyrromethene difluoride (BODIPYTM
)
(Johnson et al., 1991) fluorophore linked to the
long-chain base of naturally occurring (2S,3R)-␤-
lactosylceramide (LacCer) via the ␻ end of a N-
pentanoyl moiety (compound a in Fig. 1) has been used
to examine the intracellular trafficking of this and other
glycosphingolipids (GSLs) in normal and disease cell
types (Pagano et al., 2000). This GSL was localized in
lysosomes of a diseased cell type, but was observed at
the Golgi complex in normal fibroblasts (Chen et al.,
1998). (2S,3R)-C5-BODIPYTM-LacCer (which is avail-
able commercially) and a synthetic analogue bearing
2. Experimental
a maltosyl polar head group (2S,3R)-C5-BODIPYTM
-
2.1. Materials and analytical procedures
2.1.1. Chemicals
∗
Corresponding author. Tel.: +1 718 997 3279;
The sources of the chemicals were as follows:
fax: +1 718 997 3349.
E-mail address: robert.bittman@qc.cuny.edu (R. Bittman).
BODIPYTM-C5-N-hydroxysuccinimidoyl (NHS) ester,
0009-3084/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2006.03.001

Y. Liu, R. Bittman / Chemistry and Physics of Lipids 142 (2006) 58–69
59
Fig. 1. Structures of (a) (2S,3R) (or d-erythro); (b) (2R,3R) (or d-threo); (c) (2R,3S) (or l-erythro); and (d) (2S,3S) (or l-threo)-BODIPYTM-LacCer.
Invitrogen/Molecular Probes (Eugene, OR); N-Boc-d-
serine and diisobutylaluminum hydride (DIBAL-H, a
20 wt.% solution in toluene), Acros (Morris Plains, NJ);
l-threo-sphingosine, Avanti Polar Lipids (Alabaster,
AL); 1-pentadecyne, p-toluenesulfonic acid monohy-
drate (p-TsOH), and sodium bis(2-methoxyethoxy)alu-
minum hydride (Red-Al, a 70%, w/w solution in tol-
uene), Alfa Aesar/Lancaster (Pelham, NH); ␤-d-lactosyl
octaacetate, triphenylphosphine, trichloroacetonitrile,
tert-butyldiphenylsilyl chloride (TBDPSCl), hydrazine
acetate, benzoic anhydride, BF3·OEt2, imidazole,
4-(dimethylamino)pyridine (DMAP), and (n-Bu)4NF
(TBAF), Sigma–Aldrich. Trifluoromethanesulfonyl
azide (TfN3) was prepared according to Vasella et al.
(1991). Hepta-O-acetyllactosyl-1-trichloroacetimidate
(compound 13) was synthesized from per-O-ace-
tyllactose as described (Amvam-Zollo and Sinay, 1986).
Molecular sieves (300AW) were dried for 5 h at 150 ◦C
and stored under vacuum over P2O5.
pounds were visualized by charring with 10% H2SO4 in
EtOH or by UV light. Column chromatography was car-
ried out with silica gel 60 (230–400 mesh) using the elu-
tion solvents indicated in the text. Suspended silica gel
was removed by filtration through an Osmonics Cameo
filter (Fisher Scientific, Pittsburgh, PA). The 1H and 13
C
NMRspectrawererecordedat400and100 MHz, respec-
tively, and were referenced to the residual CHCl3 at δ
7.24 (1H) and the central line of CDCl3 at δ 77.0 ppm
(13C). Optical rotations were measured on a digital
polarimeter at room temperature in the solvents stated.
2.2. Synthesis
2.2.1. N-[(1,1-Dimethylethoxy)carbonyl]-d-serine
methyl ester (2)
To a cold solution of N-Boc-d-serine (compound 1 in
Scheme 1, 3.0 g, 14.6 mmol) in DMF (20 ml) was added
potassium carbonate (2.28 g, 16.5 mmol). After the mix-
ture was stirred for 10 min in an ice-water bath, methyl
iodide (1.88 ml, 4.26 g, 30 mmol) was added to the
white suspension, and stirring was continued at 0 ◦C for
30 min, whereupon the mixture solidified. The reaction
mixture was warmed to room temperature and stirred for
an additional hour. The reaction mixture was filtered by
suction and the filtrate was partitioned between EtOAc
(30 ml)andwater(30 ml). Theorganicphasewaswashed
withbrine(2 × 30 ml), dried(Na2SO4), filtered, andcon-
2.1.2. General methods
Air- and moisture-sensitive reactions were carried
out under nitrogen in flame-dried glassware. THF and
toluene were distilled from sodium/benzophenone and
dichloromethane was distilled from calcium hydride
prior to use. DMF was dried over calcium hydride. TLC
was performed using aluminum-backed or glass-backed
silica gel 60 F254 plates (0.25-mm thick), and the com-

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Y. Liu, R. Bittman / Chemistry and Physics of Lipids 142 (2006) 58–69
centrated to give 2.76 g (86%) of compound 2 as a pale
amber oil, which was used without further purification.
−78 ◦C. The reaction mixture was allowed to warm to
room temperature overnight. The reaction was quenched
by the addition of saturated aqueous NH4Cl solution
(20 ml) at −20 ◦C. After dilution with water (20 ml),
the aqueous layer was separated and extracted with
Et2O (2 × 20 ml). The combined organic layers were
washed with brine (10 ml), dried (Na2SO4), filtered, and
concentrated. The residue was purified by chromatog-
raphy (elution with hexane/EtOAc 4:1) to give 814 mg
(62%) of compound 5; Rf 0.45 (hexane/EtOAc 4:1); 1H
NMR (CDCl3) δ 4.37–4.36 (m, 1H), 2.46–2.45 (m, 1H),
1.89–1.88 (m, 1H), 1.74–1.68 (m, 2H), 1.47–1.45 (m,
2H), 1.40–1.26 (m, 37H), 0.88 (t, 3H, J = 7.2 Hz); 13C
NMR (CDCl3) δ 82.7, 70.4, 60.0, 35.3, 29.5, 27.3, 27.2,
27.16, 27.12, 27.0, 26.9, 22.6, 20.3, 11.7.
2.2.2. 3-(1,1-Dimethylethyl) 4-methyl-(R)-2,2-
dimethyl-3,4-oxazolidinedicarboxylate (3)
To a 250 ml round-bottomed flask were added a solu-
tion of compound 2 (2.76 g, 12.5 mmol) in benzene
(75 ml), 2,2-dimethoxypropane(DMP, 2.61 g, 25 mmol),
and p-TsOH (33 mg, 0.18 mmol). The colorless solution
was heated under reflux for 1 h, then slowly distilled
until a volume of 65 ml was collected over 30 min. The
cooled, amber solution was partitioned between satu-
rated NaHCO3 solution (20 ml) and Et2O (2 × 50 ml).
The organic layer was washed with brine (20 ml), then
dried (Na2SO4), filtered, and concentrated to give crude
product 3 as an amber oil. The material was vacuum dis-
tilledtogive2.68 g(80%)ofcompound3asapaleyellow
liquid, bp 101–102 ◦C (2 mm Hg); 1H NMR (CDCl3) δ
4.48–4.37 (m, 1H), 4.18–4.12 (m, 1H), 1.67–1.64 (m,
3H), 1.53–1.41 (m, 15H); 13C NMR (CDCl3) δ 171.4,
151.2, 95.1, 80.4, 66.3, 59.3, 52.4, 28.4, 28.3, 27.3, 26.0,
25.2, 25.0, 24.4.
2.2.5. tert-Butyl (1R,2R)-N-[2-hydroxy-1-
(hydroxymethyl)-3-heptadecynyl]-carbamate (6)
To a solution of 0.70 g (1.60 mmol) of compound 5 in
10 ml of MeOH was added 0.50 g of Amberlyst 15 resin.
After the heterogeneous mixture was stirred at room
temperature for 48 h, the mixture was filtered through
a Celite pad, and the filtrate was concentrated. Purifi-
cation by chromatography (elution with hexane/EtOAc
1:1) gave 480 mg (75%) of compound 6 as a white solid;
2.2.3. 1,1-Dimethylethyl (R)-4-formyl-2,2-dimethyl-
3-oxazolidinecarboxylate (4)
1
A solution of compound 3 (2.68 g, 10 mmol) in
toluene (25 ml) was cooled to −78 ◦C under nitrogen.
To the cooled solution, was slowly added a solution
of 1.5 M DIBAL-H in toluene (12 ml, 18 mmol). The
reaction mixture was stirred for 2 h at −78 ◦C, and was
then quenched by slowly adding 5 ml of cold MeOH.
The resulting white emulsion was slowly poured into
50 ml of ice-cold 1 N HCl with swirling over 15 min,
and the aqueous mixture was extracted with EtOAc
(3 × 50 ml). The combined organic layers were washed
with brine (50 ml), dried (Na2SO4), filtered, and con-
centrated to give the crude product as a colorless oil.
The material was vacuum distilled to give 1.72 g (75%)
of compound 4 as a colorless liquid, bp 83–88 ◦C (1.0–
1.4 mm Hg).
Rf 0.52 (hexane/EtOAc 1:1); H NMR (CDCl3) δ 5.18
(m, 1H), 4.60 (s, 1H), 3.83–3.77 (m, 3H), 3.35 (s, 1H),
2.91 (s, 1H), 2.22–2.18 (m, 2H), 1.52–1.30 (m, 31H),
0.88 (t, 3H, J = 7.2 Hz); 13C NMR (CDCl3) δ 156.4, 87.4,
80.0, 78.1, 63.6, 62.9, 55.9, 31.9, 29.7, 29.6, 29.5, 29.3,
29.1, 28.9, 28.6, 28.3, 28.1, 22.7, 18.7, 14.1.
2.2.6. tert-Butyl (3E,1R,2R)-N-[2-hydroxy-1-
(hydroxymethyl)-3-heptadecenyl]-carbamate (7)
To a solution of 440 mg (1.0 mmol) of compound
6 in dry Et2O (20 ml) was added dropwise 3.0 ml
(10.5 mmol) of Red-Al (a 3.5 M solution in toluene)
at 0 ◦C under nitrogen. After the reaction mixture was
stirred at room temperature for 24 h, the reaction was
quenched by the slow addition of 3 ml of MeOH at 0 ◦C.
The product was extracted with EtOAc (3 × 20 ml), and
the combined organic layers were washed with brine
(10 ml), dried (Na2SO4), filtered, and concentrated. The
residue was purified by chromatography (elution with
hexane/EtOAc 1:1) to give 264 mg (60%) of compound
7 as a white solid; Rf 0.38 (hexane/EtOAc 1:1); 1H NMR
(CDCl3) δ 5.75 (m, 1H), 5.53 (m, 1H), 5.18 (m, 1H), 4.33
(s, 1H), 3.80–3.55 (m, 3H), 2.71 (s, 2H), 2.05 (m, 2H),
1.45–1.05 (m, 31H), 0.88 (t, 3H, J = 6.8 Hz); 13C NMR
(CDCl3) δ 156.6, 134.0, 129.0, 79.8, 73.5, 64.4, 55.5,
32.3, 31.9, 29.69, 29.66, 29.6, 29.5, 29.4, 29.2, 29.1,
28.4, 22.7, 14.1.
2.2.4. tert-Butyl (4R,1ꢀR)-2,2-dimethyl-4-
(1ꢀ-hydroxyhexadec-2ꢀ-ynyl)oxazolidione-3-
carboxylate (5)
n-Butyllithium (2.5 M in hexane, 2.0 ml, 5.0 mmol)
was added dropwise to a solution of 1-pentadecyne
(832 mg, 4.0 mmol) in dry Et2O (20 ml) at −20 ◦C (see
Scheme 2). After the white suspension was stirred at
−20 ◦C for 1 h, anhydrous ZnBr2 (1.2 g, 5.0 mmol) was
added at 0 ◦C, with stirring for 1 h at 0 ◦C and 1 h at
room temperature. A solution of compound 4 (690 mg,
3.0 mmol) in dry Et2O (10 ml) was added dropwise at

Y. Liu, R. Bittman / Chemistry and Physics of Lipids 142 (2006) 58–69
61
2.2.10. (1ꢀR,1R,2E)-Benzoic acid 1-[1ꢀ-azido-2ꢀ-
(tert-butyldiphenylsilyloxy)-ethyl]-hexadec-2-enyl
ester (11)
2.2.7. d-threo-Sphingosine (8)
A solution of 240 mg (0.60 mmol) of compound 7 in
5 ml of 1 M HCl and 5 ml of THF was heated at 70 ◦C
with stirring for 8 h under nitrogen. The reaction mixture
was cooled to room temperature and neutralized with
saturated aqueous NaHCO3 solution (5 ml). The product
was extracted with EtOAc (3 × 20 ml), and the combined
organic layer was washed with brine, dried (Na2SO4),
filtered, and concentrated to give 140 mg (78%) of com-
pound 8 as a white powder, which was used without
further purification.
To a solution of compound 10 (65 mg, 0.115 mmol)
in 10 ml of dry CH2Cl2 was added DMAP (50 mg,
0.40 mmol), followed by the dropwise addition of a solu-
tion of benzoic anhydride (45 mg, 0.20 mmol) in 5 ml of
CH2Cl2 at 0 ◦C. The reaction mixture was allowed to
warm to room temperature and stirred overnight. The
solvent was removed under reduced pressure, and the
residue was purified by chromatography (elution with
hexane/EtOAc 19:1) to give 70 mg (92%) of compound
11; Rf 0.80 (hexane/EtOAc 4:1); [␣]2D5 −4.82◦ (c 3.90,
2.2.8. (2R,3R,4E)-2-Azido-octadec-4-ene-1,3-diol
(9)
1
CHCl3); H NMR (CDCl3) δ 8.00 (m, 2H), 7.68–7.60
(m, 4H), 7.55 (m, 1H), 7.45–7.28 (m, 8H), 5.87 (m, 1H),
5.63 (m, 1H), 5.43 (m, 1H), 4.12 (m, 1H), 3.80 (m, 2H),
3.62 (m, 1H), 2.00 (m, 2H), 1.40–1.01 (m, 31H), 0.88
(t, 3H, J = 6.8 Hz); 13C NMR (CDCl3) δ 165.3, 137.7,
135.9, 133.3, 133.2, 133.1, 132.9, 132.8, 74.1, 65.9, 63.3,
32.3, 32.0, 29.7, 29.6, 29.5, 29.4, 29.2, 29.1, 28.9, 28.7,
26.9, 26.7, 22.7, 19.2, 14.1.
Dichloromethane (10 ml) and DMAP (150 mg,
1.23 mmol) were added to compound 8 (120 mg,
0.40 mmol), followed by dropwise addition of TfN3
in CH2Cl2 (0.4 M solution, 10 ml, 4.0 mmol) (see
Scheme3). Thereactionmixturewasstirredatroomtem-
perature for 24 h, and then concentrated under reduced
pressure. The residue was purified by chromatography
(elution with hexane/EtOAc 1:1) to give 60 mg (46%) of
azido diol 9; Rf 0.60 (hexane/EtOAc 1:1); [␣]2D5 −3.05◦
(c 2.59, CHCl3); 1H NMR (CDCl3) δ 5.78 (m, 1H), 5.52
(m, 1H), 4.21 (m, 1H), 3.80 (m, 1H), 3.72 (m, 1H), 2.42
(s, 2H), 2.07 (m, 2H), 1.45–1.18 (m, 22H), 0.88 (t, 3H,
J = 6.8 Hz); 13C NMR (CDCl3) δ 135.5, 128.2, 73.5,
67.6, 62.9, 32.3, 31.9, 29.7, 29.6, 29.5, 29.47, 29.36,
29.2, 29.1, 28.9, 22.7, 14.1.
2.2.11. (1ꢀR,1R,2E)-Benzoic acid
1-(1ꢀ-azido-2ꢀ-hydroxyethyl)hexadec-2-
enyl ester (12)
To a solution of compound 11 (68 mg, 0.10 mmol)
and 50 mg (0.72 mmol) of imidazole in 5 ml of dry THF
was added TBAF (0.2 ml, 0.20 mmol, a 1 M solution in
THF) at −23 ◦C. The reaction mixture was stirred at
−23 ◦Cfor3 h, andwasthenquicklypassed(tominimize
benzoyl migration) through a silica gel column that was
prewashed with cold elution solvent (elution with hex-
ane/EtOAc 4:1) to give 26 mg (60%) of compound 12; Rf
0.35 (hexane/EtOAc 4:1); [␣]2D5 −8.34◦ (c 0.42, CHCl3).
1H NMR (CDCl3) δ 8.08 (m, 2H), 7.72–7.37 (m, 3H),
5.98 (m, 1H), 5.65 (m, 1H), 5.56 (m, 1H), 3.70 (m, 3H),
2.17 (m, 1H), 2.06 (m, 2H), 1.53–1.24 (m, 22H), 0.88
(t, 3H, J = 6.8 Hz); 13C NMR (CDCl3) δ 165.8, 138.1,
134.8, 133.4, 129.9, 129.6, 128.5, 127.7, 124.0, 74.7,
66.2, 61.7, 32.3, 31.9, 29.7, 29.6, 29.42, 29.37, 29.2,
28.7, 26.6, 22.7, 14.1.
2.2.9. (2R,3R,4E)-2-Azido-1-(tert-
butyldiphenylsilanyloxy)-octadec-4-en-
3-ol (10)
A solution of TBDPSCl (50 mg, 0.18 mmol) and imi-
dazole (25 mg, 0.36 mmol) in 10 ml of CH2Cl2 was
stirred at room temperature for 1 h. A solution of com-
pound 9 (55 mg, 0.167 mmol) in 5 ml of CH2Cl2 was
added, and the reaction mixture was stirred overnight.
The solvent was removed under reduced pressure, and
the residue was purified by chromatography (elution
with hexane/EtOAc 4:1) to give 68 mg (91%) of com-
pound 10; Rf 0.57 (hexane/EtOAc; 4:1); [␣]2D5 −11.92◦
(c 2.50, CHCl3); 1H NMR (CDCl3) δ 7.68–7.66 (m, 4H),
7.45–7.34 (m, 6H), 5.70 (m, 1H), 5.40 (m, 1H), 4.12
(m, 1H), 3.80 (m, 2H), 3.41 (m, 1H), 2.16 (s, 1H), 1.99
(m, 2H), 1.40–1.01 (m, 31H), 0.88 (t, 3H, J = 6.8 Hz);
13C NMR (CDCl3) δ 136.1, 135.63, 135.59, 135.2,
132.9, 132.8, 129.9, 129.7, 129.5, 128.3, 127.8, 127.7,
127.6, 127.3, 72.3, 67.9, 64.6, 32.3, 31.9, 29.70, 29.68,
29.6, 29.5, 29.4, 29.2, 29.1, 28.9, 28.4, 26.8, 22.7, 19.2,
14.1.
2.2.12. (1ꢀR,1R,2E)-Benzoic acid 1-[1ꢀ-azido-2ꢀ-(β-
hepta-O-acetyllactosyl)-ethyl]-hexadec-2-
enyl ester (14)
A mixture of 53 mg (0.068 mmol) of trichloroacet-
imidate 13 (see Scheme 4), 25 mg (0.058 mmol) of com-
pound 12, 200 mg of molecular sieves 300AW, and 5 ml
of CH2Cl2 was stirred at room temperature for 1 h.
A solution of BF3·Et2O (40 ␮l, 0.32 mmol) in 5 ml of
CH2Cl2 was added, and the reaction mixture was stirred

62
Y. Liu, R. Bittman / Chemistry and Physics of Lipids 142 (2006) 58–69
overnight. The solvent was removed under reduced pres-
sure, and the residue was purified by chromatography
(elution with hexane/EtOAc 1:1) to give 40 mg (66%)
of compound 14; Rf 0.55 (hexane/EtOAc 1:1); [␣]D25
THF (10 ml) at −78 ◦C. The reaction mixture was stirred
for 1 h at −78 ◦C, allowed to warm to −20 ◦C within
2 h, and quenched by the addition of saturated aqueous
NH4Cl solution (20 ml). The mixture was diluted with
water (20 ml), and the aqueous layer was separated and
extracted with Et2O (3 × 20 ml). The combined organic
layers were washed with 0.5 N HCl (2 × 10 ml) and brine
(10 ml), dried (Na2SO4), filtered, and concentrated. The
residue was purified by chromatography (elution with
hexane/EtOAc 4:1) to give 788 mg (60%) of compound
−13.1◦ (c 2.0, CHCl3); H NMR (CDCl3) δ 8.07 (m,
1
2H), 7.62–7.41 (m, 3H), 5.88 (m, 1H), 5.60 (m, 1H),
5.48 (m, 1H), 5.35 (m, 1H), 5.24–5.08 (m, 2H), 4.93 (m,
2H), 4.50 (m, 3H), 4.11 (m, 4H), 3.86 (m, 3H), 3.62 (m,
2H), 2.30–1.90 (m, 21H), 1.80–1.00 (m, 24H), 0.89 (t,
3H, J = 6.8 Hz).
1
17; Rf 0.48 (hexane/EtOAc 4:1); H NMR (CDCl3) δ
2.2.13. (2R,3R,4E)-2-Azido-1-(β-hepta-O-
acetyllactosyl)-octadec-4-en-3-ol (15)
4.74 (m, 1H), 4.51 (m, 1H), 4.10 (m, 2H), 3.90 (s, 1H),
2.19 (m, 2H), 1.65–1.45 (m, 15H), 1.40–1.20 (m, 22H),
0.88 (t, 3H, J = 6.8 Hz); 13C NMR (CDCl3) δ 152.1,
92.9, 84.6, 79.2, 75.9, 63.1, 62.1, 60.9, 29.9, 27.7, 27.6,
27.5, 27.4, 27.1, 26.9, 26.6, 26.4, 26.0, 23.8, 23.4, 21.0,
20.7,16.8, 12.1.
A solution of 6 mg (0.26 mmol) of sodium in 1 ml
of MeOH was added to 38 mg (0.036 mmol) of com-
pound 14. The reaction mixture was stirred for 6 h,
the solvent was removed under reduced pressure, and
the residue was purified by chromatography (elution
with MeOH/CHCl3 3:7) to give 16 mg (68%) of com-
pound 15; Rf 0.40 (MeOH/CHCl3 3:7); [␣]2D5 −10.6◦ (c
0.80, CHCl3/MeOH 1:1); 1H NMR (CDCl3/CD3OD) δ
5.58–5.30 (m, 2H), 5.03 (m, 1H), 4.15–2.95 (m, 17H),
1.00 (m, 24H), 0.89 (t, 3H, J = 6.8 Hz); HRMS (ESI)
calcd for C30H55N3O12Na (M + Na)+ m/z 672.3683,
found 672.3666.
2.2.16. tert-Butyl (1R,2S)-N-[2-hydroxy-1-
(hydroxymethyl)-3-heptadecynyl]-
carbamate (18)
To a solution of 0.50 g (1.14 mmol) of compound 17
in 10 ml of MeOH was added 0.40 g of Amberlyst 15
resin, and the heterogeneous mixture was stirred at room
temperature for 48 h. The mixture was filtered through
a Celite pad, and the filtrate was concentrated. Purifi-
cation by chromatography (elution with hexane/EtOAc
1:1) afforded 329 mg (73%) of compound 18 as a white
solid, which was used without further purification; Rf
0.55 (hexane/EtOAc 1:1).
2.2.14. C5-BODIPYTM-d-threo-LacCer (16)
BODIPYTM-C5-NHS (5 mg, 0.020 mmol), triph-
enylphosphine (6 mg, 0.023 mmol), 2.7 ml of THF, and
0.3 ml of water were added to 13 mg (0.020 mmol)
of compound 15. After the reaction, mixture was
stirred overnight at room temperature, the solvents were
removed under reduced pressure, and the residue was
purified by chromatography (elution with MeOH/CHCl3
1:1) to give 7 mg (38%) of compound 16; Rf
0.35 (MeOH/CHCl3 1:4); 1H NMR (CDCl3/CD3OD)
δ 7.60 (m, 2H), 7.07 (s, 1H), 6.87 (s, 1H), 6.23 (m,
1H), 6.04 (m, 1H), 3.83–2.15 (m, 18H), 1.70–0.70
(m, 35H); LRMS (APCI, negative-ion mode) calcd
for C46H74BClF2N3O13 (M + 35Cl)− m/z 960.5, found
960.5; HRMS (EI) calcd for C46H75N3O13F2B (MH+
of the boron-10 isotope) m/z 925.5397, found 925.5408.
2.2.17. tert-Butyl (3E,1R,2S)-N-[2-hydroxy-1-
(hydroxymethyl)-3-heptadecenyl]-
carbamate (19)
To a solution of 300 mg (0.76 mmol) of compound
18 in dry Et2O (20 ml) was added dropwise 2.0 ml
(7.0 mmol) of Red-Al (a 3.5 M solution in toluene) at
0 ◦C under nitrogen. After the reaction mixture was
stirred at room temperature for 24 h, the reaction was
quenched by the slow addition of 3 ml of MeOH at 0 ◦C.
The product was extracted with EtOAc (3 × 20 ml), and
the combined organic layers were washed with brine
(10 ml), dried (Na2SO4), filtered, and concentrated. The
residue was purified by chromatography (elution with
hexane/EtOAc 1:1) to give 181 mg (60%) of compound
2.2.15. tert-Butyl (4R,1ꢀS)-2,2-dimethyl-4-(1ꢀ-
hydroxyhexadec-2ꢀ-ynyl)oxazolidione-3-
carboxylate (17)
1
19 as a white solid; Rf 0.40 (hexane/EtOAc 1:1); H
n-Butyllithium (2.5 M in hexane, 2.0 ml, 5.0 mmol)
was added dropwise to a solution of 1-pentadecyne
(832 mg, 4.0 mmol) in dry THF (20 ml) at −20 ◦C (see
Scheme 5). After the mixture was stirred at −20 ◦C for
2 h, HMPA (0.73 ml, 5.0 mmol) was added, followed by
a solution of compound 4 (690 mg, 3.0 mmol) in dry
NMR (CDCl3) δ 5.78 (m, 1H), 5.52 (m, 1H), 5.33 (m,
1H), 4.28 (m, 1H), 3.90 (m, 1H), 3.70 (m, 1H), 3.60 (s,
1H), 3.05 (s, 2H), 2.05 (m, 2H), 1.50–1.30 (m, 31H), 0.88
(t, 3H, J = 6.8 Hz); 13C NMR (CDCl3) δ 156.3, 134.1,
129.0, 79.8, 74.7, 62.61, 55.47, 32.3, 31.9, 31.7, 29.70,
29.67, 29.6, 29.5, 29.4, 29.3, 29.2, 28.4, 22.7, 14.1.

Y. Liu, R. Bittman / Chemistry and Physics of Lipids 142 (2006) 58–69
63
2.2.21. (1ꢀR,1S,2E)-Benzoic acid 1-[1ꢀ-azido-2ꢀ-
(tert-butyldiphenylsilanyloxy)-ethyl]-hexadec-2-enyl
ester (23)
2.2.18. l-erythro-Sphingosine (20)
A solution of 160 mg (0.40 mmol) of compound 19
in 5 ml of 1 M HCl and 5 ml of THF was heated at 70 ◦C
with stirring for 8 h under nitrogen. The reaction mixture
was cooled to room temperature and neutralized with
saturated aqueous NaHCO3 solution (5 ml). The product
was extracted with EtOAc (3 × 20 ml), and the combined
organiclayerwaswashedwithbrine, dried(Na2SO4), fil-
tered, and concentrated to give 90 mg (75%) compound
20 as a white powder, which was used without further
purification.
Compound 23 was prepared by the method used to
synthesize compound 11. DMAP (40 mg, 0.32 mmol)
was added to a solution of compound 22 (50 mg,
0.089 mmol) in 10 ml of dry CH2Cl2, followed by the
dropwise addition of a solution of benzoic anhydride
(34 mg, 0.15 mmol) in 5 ml of CH2Cl2 at 0 ◦C. After
the reaction mixture was stirred overnight room tem-
perature, concentration gave a residue that was puri-
fied by chromatography (elution with hexane/EtOAc
19:1), affording 53 mg (89%) of compound 23; Rf 0.85
(hexane/EtOAc 4:1); [␣]2D5 +11.6◦ (c 0.71, CHCl3); 1H
NMR (CDCl3) δ 8.01 (m, 2H), 7.68–7.50 (m, 5H),
7.48–7.28 (m, 8H), 5.90 (m, 1H), 5.68 (m, 1H), 5.50
(m, 1H), 3.90–3.70 (m, 4H), 2.00 (m, 2H), 1.50–1.01
(m, 31H), 0.88 (t, 3H, J = 6.8 Hz); 13C NMR (CDCl3)
δ 165.2, 135.6, 135.4, 133.1, 132.9, 132.7, 130.1,
129.8, 129.7. 128.4, 127.8, 123.2, 74.1, 65.9, 63.3,
31.9, 29.7, 29.6, 29.4, 19.1, 28.7, 26.7, 22.7, 19.2,
14.1.
2.2.19. (2R,3S,4E)-2-Azido-octadec-4-ene-1,3-diol
(21)
The diazo transfer reaction was carried out as
described for the synthesis of compound 9. To compound
20 (80 mg, 0.27 mmol) were added CH2Cl2 (10 ml) and
DMAP (100 mg, 0.82 mmol), followed by the dropwise
addition of a solution of TfN3 in CH2Cl2 (0.4 M solu-
tion, 7.0 ml, 2.8 mmol) (see Scheme 6), with stirring at
room temperature for 24 h. Concentration gave a residue
that was purified by chromatography (elution with hex-
ane/EtOAc 1:1), affording 40 mg (46%) of azido diol
21; Rf 0.50 (hexane/EtOAc 1:1); [␣]2D5 +25.2◦ (c 0.80,
2.2.22. (1ꢀR,1S,2E)-Benzoic acid
1
1-(1ꢀ-azido-2ꢀ-hydroxyethyl)hexadec-2-enyl ester
(24)
CHCl3); H NMR (CDCl3) δ 5.80 (m, 1H), 5.53 (m,
1H), 4.25 (m, 1H), 3.78 (m, 2H), 3.50 (m, 1H), 2.23
(s, 2H), 2.08 (m, 2H), 1.40–1.20 (m, 22H), 0.88 (t, 3H,
J = 7.2 Hz); 13C NMR (CDCl3) δ 136.0, 128.0, 73.8,
66.8, 62.6, 32.3, 31.9, 29.69, 29.66, 29.6, 29.5, 29.4,
29.2, 28.9, 22.7, 14.1.
The desilylation reaction was carried out as described
for the preparation of compound 11 (2 equiv of TBAF,
∼7 equiv of imidazole, dry THF, −23 ◦C). The reac-
tion mixture was stirred at −23 ◦C for 3 h, and then
was quickly passed through a silica gel column that
was prewashed with cold elution solvent (elution with
hexane/EtOAc 4:1) to give 19 mg (59%) of compound
24; Rf 0.30 (hexane/EtOAc 4:1); [␣]2D5 +38.2◦ (c 0.40,
CHCl3);1H NMR (CDCl3) δ 8.06 (m, 2H), 7.60–7.44
(m, 3H), 5.94 (m, 1H), 5.61 (m, 2H), 3.80 (m, 2H),
3.64 (m, 1H), 2.10 (m, 3H), 1.50–1.24 (m, 22H), 0.88
(t, 3H, J = 6.8 Hz); 13C NMR (CDCl3) δ 165.5, 138.9,
133.4, 129.8, 129.7, 128.5, 123.3, 74.6, 66.2, 62.0,
32.4, 31.9, 29.7, 29.6, 29.43, 29.37, 29.2, 28.7, 22.7,
14.1.
2.2.20. (2R,3S,4E)-2-Azido-1-(tert-
butyldiphenylsilyloxy)-octadec-4-en-3-ol (22)
The silylation reaction was carried out as described
for the preparation of compound 10. A solution of com-
pound 21 (39 mg, 0.12 mmol) in 5 ml of CH2Cl2 was
added to TBDPSCl (35 mg, 0.13 mmol) and imidazole
(18 mg, 0.26 mmol) in 10 ml of CH2Cl2. The reaction
mixture was stirred overnight, the solvent was removed
under reduced pressure, and the residue was purified by
chromatography(elutionwithhexane/EtOAc4:1)togive
56 mg (83%) of compound 22; Rf 0.60 (hexane/EtOAc
4:1); [␣]2D5 +10.7◦ (c 0.80, CHCl3); H NMR (CDCl3)
1
2.2.23. (1ꢀR,1S,2E)-Benzoic acid 1-[1ꢀ-azido-2ꢀ-(β-
heptaacetyllactosyl)-ethyl]-hexadec-2-
enyl ester (25)
A mixture of 53 mg (0.068 mmol) of trichloroacetim-
idate 13, 19 mg (0.044 mmol) of compound 24, 100 mg
of molecular sieves 300AW, and 5 ml of CH2Cl2 was
stirred at room temperature for 1 h (see Scheme 7). Then
a solution of BF3·OEt2 (40 ␮l, 0.32 mmol) in 5 ml of
CH2Cl2 was added, and the reaction mixture was stirred
δ 7.68–7.66 (m, 4H), 7.45–7.34 (m, 6H), 5.72 (m, 1H),
5.43 (m, 1H), 4.22 (m, 1H), 3.79 (m, 2H), 3.51 (m, 1H),
2.11 (s, 1H), 2.01 (m, 2H), 1.36–1.01 (m, 31H), 0.88
(t, 3H, J = 6.8 Hz); 13C NMR (CDCl3) δ 134.1, 134.0,
133.7, 133.6, 133.5, 131.9, 131.7, 131.2, 130.9, 128.0,
127.8, 127.6, 125.9, 125.8, 70.9, 65.0, 62.2, 50.3, 30.4,
30.0, 27.8, 27.7, 27.6, 27.5, 27.3, 27.1, 25.0, 24.9, 24.8,
20.8, 17.3, 17.2, 12.2.

64
Y. Liu, R. Bittman / Chemistry and Physics of Lipids 142 (2006) 58–69
overnight. The solvent was removed under reduced pres-
sure, and the residue was purified by chromatography
(elution with hexane/EtOAc 1:1) to give 25 mg (54%)
of compound 25; Rf 0.50 (hexane/EtOAc 1:1); 1H NMR
(CDCl3) δ 7.97 (m, 2H), 7.56–7.38 (m, 3H), 5.80 (m,
1H), 5.63 (m, 1H), 5.50 (m, 1H), 5.40 (m, 1H), 5.28 (m,
1H), 5.10 (m, 2H), 4.89 (m, 2H), 4.45 (m, 3H), 4.05 (m,
4H), 3.80 (m, 3H), 3.55 (m, 2H), 2.10–1.86 (m, 21H),
1.35–1.12 (m, 24H), 0.80 (t, 3H, J = 6.8 Hz).
2.2.27. 2-Azido-1-(tert-butyldiphenylsilanyloxy)-
l-threo-sphingosine (30)
Compound 30 was prepared by the method used to
synthesize compound 10. A solution of compound 29
(26 mg, 0.080 mmol) in 5 ml of CH2Cl2 was added to
TBDPSCl (23 mg, 0.084 mmol) and imidazole (12 mg,
0.17 mmol) in 5 ml of CH2Cl2. The reaction mixture
was stirred overnight, the solvent was removed, and the
residue was purified by chromatography (elution with
hexane/EtOAc from 9:1 to 4:1) to give 39 mg (91%) of
1
compound 30; Rf 0.77 (hexane/EtOAc 4:1); H NMR
2.2.24. (2R,3R,4E)-2-Azido-1-(β-hepta-O-
(CDCl3) δ 7.71 (m, 4H), 7.46 (m, 6H), 5.64 (dt, 1H,
J = 10.8, 7.2 Hz), 5.42 (dd, 1H, J = 10.8, 8.8 Hz), 4.61
(m, 1H), 3.84 (m, 2H), 3.56 (m, 1H), 2.01 (m, 2H), 1.61
(s, 1H), 1.40–1.06 (m, 31H), 0.91 (t, 3H, J = 6.8 Hz);
13C NMR (CDCl3) δ 135.8, 135.6, 134.6, 129.9, 129.1,
127.9, 127.5, 67.5, 66.0, 64.1, 32.0, 29.7, 29.4, 28.0,
26.9, 26.8, 22.7, 19.1, 14.1.
acetyllactosyl)-octadec-4-en-3-ol (26)
A solution of 5.0 mg (0.22 mmol) of sodium in 1 ml of
MeOH was added to 21 mg (0.020 mmol) of compound
25. After the reaction mixture was stirred for 6 h, the
solvent was removed to give 8 mg (62%) of compound
26. HRMS (ESI) calcd for C30H55N3O12Na (M + Na)+
m/z 672.3683, found 672.3682.
2.2.28. 2-Azido-3-benzoic acid-1-(tert-
butyldiphenylsilanyloxy)-l-threo-
2.2.25. C5-BODIPYTM-l-erythro-LacCer (27)
A mixture of 8 mg (0.012 mmol) of compound
26, BODIPYTM-C5-NHS (5.0 mg, 0.020 mmol), triph-
enylphosphine (6.0 mg, 0.023 mmol), 2.7 ml of THF,
and 0.3 ml of water was stirred overnight at room
temperature. Removal of the solvents gave a residue
that was purified by chromatography (elution with
MeOH/CHCl3 1:1), affording 4 mg (36%) of com-
pound 27; Rf 0.38 (MeOH/CHCl3 1:4); 1H NMR
(CDCl3/CD3OD) δ 7.78–6.04 (m, 4H), 6.23 (m, 1H),
6.04 (m, 1H), 3.83–2.05 (m, 18H), 1.70–0.70 (m,
35H); LRMS (APCI, negative-ion mode) calcd for
C46H74BClF2N3O13 (M + 35Cl)− m/z 960.5, found
960.5; HRMS (EI) calcd for C46H75N3O13F2B
(MH+ of the boron-10 isotope) m/z 925.5397, found
925.5409.
sphingosine (31)
To a solution of compound 30 (38 mg, 0.067 mmol)
in 5 ml of dry CH2Cl2 was added DMAP (25 mg,
0.20 mmol), followed by the dropwise addition of a solu-
tion of benzoic anhydride (18 mg, 0.080 mmol) in 5 ml
of CH2Cl2 at 0 ◦C. The reaction mixture was stirred
overnight, the solvent was removed, and the residue
was purified by chromatography (elution with hexane,
then with hexane/EtOAc 19:1) to give 38 mg (85%) of
1
compound 31; Rf 0.90 (hexane/EtOAc 4:1); H NMR
(CDCl3) δ 8.04 (m, 2H), 7.71 (m, 4H), 7.46 (m, 9H), 6.06
(m, 1H), 5.77 (dt, 1H, J = 10.8, 7.2 Hz), 5.50 (dd, 1H,
J = 10.8, 8.8 Hz), 3.82 (m, 3H), 2.25 (m, 2H), 1.50–1.10
(m, 31H), 0.93 (t, 3H, J = 6.8 Hz); 13C NMR (CDCl3) δ
162.8, 135.8, 133.5, 133.23, 133.19, 133.0, 132.4, 132.0,
130.7, 130.5, 130.4, 128.1, 127.7, 127.52, 127.51, 127.4,
127.2, 127.1, 126.1, 125.5, 125.44, 125.35, 125.1, 120.5,
67.0, 63.72, 63.67, 61.1, 29.6, 27.4, 27.34, 27.28, 27.2,
27.09, 27.06, 27.0, 25.9, 24.7, 24.5, 24.4, 23.3, 20.4,
16.8, 11.8. HRMS (ESI) calcd for C41H57N3O3SiNa
(M + Na)+ m/z 690.4067, found 690.4081.
2.2.26. 2-Azido-l-threo-sphingosine (29)
TfN3 (1 ml, 0.4 mmol, a 0.4 M solution in CH2Cl2)
was added dropwise to l-threo-sphingosine (com-
pound 28, 25 mg, 0.084 mmol) and DMAP (20 mg,
0.164 mmol) in 5 ml of CH2Cl2 (see Scheme 8). The
reaction mixture was stirred at room temperature for
24 h, and then concentrated to give a residue that was
purified by chromatography (elution with hexane/EtOAc
3:1), affording 26 mg (95%) of compound 29; Rf 0.82
(hexane/EtOAc 1:1); 1H NMR (CDCl3) δ 5.69 (dt, 1H,
J = 10.8, 7.2 Hz), 5.48 (dd, 1H, J = 10.8, 8.8 Hz), 4.62 (m,
1H), 3.81 (m, 2H), 3.52 (m, 1H), 2.13 (m, 2H), 1.45–1.20
(m, 22H), 0.91 (t, 3H, J = 7.2 Hz); 13C NMR (CDCl3) δ
136.0, 127.5, 68.2, 66.9, 62.6, 31.9, 29.69, 29.66, 29.6,
29.5, 29.4, 29.3, 28.0, 22.7, 14.1.
2.2.29. 2-Azido-3-benzoic acid-l-threo-
sphingosine (32)
TBAF (0.1 ml, 0.1 mmol, 1 M in THF) was added to
a solution of compound 31 (35 mg, 0.051 mmol) and
25 mg (0.36 mmol) of imidazole in 5 ml of dry CH2Cl2
at −23 ◦C. After being stirred at −23 ◦C for 3 h, the reac-
tion mixture was quickly passed through a silica gel col-
umn that had been prewashed with cold elution solvent.
Elution with hexane/EtOAc 4:1) afforded 14 mg (63%)

Y. Liu, R. Bittman / Chemistry and Physics of Lipids 142 (2006) 58–69
65
of compound 32; Rf 0.65 (hexane/EtOAc 4:1); 1H NMR
(CDCl3) δ 8.09 (m, 2H), 7.46 (m, 3H), 6.00 (m, 1H), 5.75
(dt, 1H, J = 10.8, 7.2 Hz), 5.52 (dd, 1H, J = 10.8, 8.8 Hz),
4.51 (m, 2H), 3.88 (m, 1H), 2.13 (m, 2H), 1.50–1.10
(m, 31H), 0.93 (t, 3H, J = 6.8 Hz); 13C NMR (CDCl3) δ
166.4, 165.6, 138.4, 136.5, 135.2, 134.8, 133.4, 129.9,
129.8, 129.7, 129.5, 128.53, 128.51, 127.7, 126.7, 122.8,
69.6, 67.3, 66.4, 65.1, 64.3, 61.9, 32.0, 29.71, 29.68,
29.6, 29.50, 29.48, 29.4, 29.3, 28.2, 28.1, 26.6, 22.7,
19.0, 14.2.
compound 33; Rf 0.45 (MeOH/CHCl3 1:4); H NMR
(CDCl3): same as for compound 27; HRMS (EI) calcd
for C46H75N3O13F2B (MH+ of the boron-10 isotope)
m/z 925.5397, found 925.5416.
1
3. Results
3.1. Retrosynthetic plan
As shown in the retrosynthetic plan (Fig. 2), the
preparation of the BODIPYTM-LacCer stereoisomers
consists of three building blocks: a 2-azido-3-benzo-
ylsphingosinederivativecomposedof thedesired config-
urations at C2 and C3, an activated BODIPYTM-linked
fatty acid, and an activated and protected lactosyl donor
(hepta-O-acetyl-␤-lactosyl-1-trichloroacetimidate)
(Amvam-Zollo and Sinay, 1986).
2.2.30. l-threo-C5-BODIPYTM-LacCer (33)
A mixture of 21 mg (0.027 mmol) of compound 32,
12 mg (0.027 mmol) of trichloroacetimidate 13, and
100 mg of molecular sieves 300AW in 5 ml of CH2Cl2
was stirred at room temperature for 1 h. A solution of
BF3·OEt2 (20 ␮l, 0.16 mmol) in 2 ml of CH2Cl2 was
added, and the reaction mixture was stirred overnight.
The solvent was removed under reduced pressure, and
theresiduewaspurifiedbychromatography(elutionwith
CHCl3, then with MeOH/CHCl3 1:9) to give 9 mg (30%)
of the glycosylation product. Alkaline methanolysis of
the acetate and benzoate ester functionalities was car-
ried out by adding a solution of 2 mg (0.08 mmol) of
sodium in 1 ml of dry MeOH, followed by stirring at
room temperature for 6 h. Dowex 50W-X8 resin (pre-
washed with 50 ml of MeOH) was added to neutralize the
reaction mixture. The reaction mixture was filtered and
3.2. Synthesis of d-threo C5-BODIPYTM-LacCer
analogue (16)
See Scheme 4.
solvent was removed under vacuum. After BODIPYTM
-
C5-NHS (3 mg, 0.012 mmol), triphenylphosphine (4 mg,
0.016 mmol), 2.7 ml of THF, and 0.3 ml of water were
added, the reaction mixture was stirred overnight at
room temperature. The solvents were removed, and the
residue was purified by chromatography (elution with
MeOH/CHCl3 from 1:9 to 1:4) to give 1.5 mg (38%) of
Scheme 1. Synthesis of (R)-Garner aldehyde (4).
Fig. 2. Retrosynthetic plan for the preparation of d-threo- and l-erythro-C5-BODIPYTM-LacCer.

66
Y. Liu, R. Bittman / Chemistry and Physics of Lipids 142 (2006) 58–69
Scheme 2. Synthesis of (2R,3R)-sphingosine (8).
Scheme 3. Synthesis of (2R,3R)-2-azido-3-benzoylsphingosine (12).
Scheme 4. Synthesis of (2R,3R)-C5-BODIPYTM-LacCer (16).

Y. Liu, R. Bittman / Chemistry and Physics of Lipids 142 (2006) 58–69
67
Scheme 5. Synthesis of (2R,3S)-sphingosine (20).
Scheme 6. Synthesis of (2R,3S)-2-azido-3-benzoylsphingosine (24).
Scheme 7. Synthesis of (2R,3S)-C5-BODIPYTM-LacCer (27).

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Y. Liu, R. Bittman / Chemistry and Physics of Lipids 142 (2006) 58–69
Scheme 8. Synthesis of l-threo-C5-BODIPYTM-LacCer (33).
3.3. Synthesis of l-erythro C5-BODIPYTM-LacCer
analogue (27)
azido sphingosine derivatives, compounds 9, 21, and
29. Silylation of the primary hydroxy group was carried
out in the presence of 2 equivalents of imidazole; after
benzoylation of the secondary hydroxy group, the desily-
lation reaction was performed at −23 ◦C (Mattjus et al.,
2002), followed by rapid elution through a cold silica gel
column, to minimize benzoyl migration, furnishing the
three stereoisomers of 2-azido-3-benzoylsphingosines:
compounds 12 (Scheme 3), 24 (Scheme 6), and
32 (Scheme 8). After BF3·OEt2-mediated lactosyla-
tion of the 2-azido-3-benzoylsphingosine stereoiso-
mers with hepta-O-acetyllactosyl trichloroacetimidate
in CH2Cl2 in the presence of molecular sieves,
base-catalyzed deprotection afforded the ␤-lactosyl-2-
azidosphingosines. Staudinger reduction of the azido
group with triphenylphosphine in aqueous THF
(Gololobov et al., 1981), followed by N-acylation with
the N-hydroxysuccinimidoyl ester of BODIPYTM-C5
and purification by column chromatography on silica
gel (elution with CHCl3/MeOH 4:1, v/v), furnished
the target unnatural BODIPYTM-LacCer stereoisomers:
compounds 16 (Scheme 4), 27 (Scheme 7), and 33
(Scheme 8). NMR spectroscopy and mass spectrometry
confirmed the structures of these analogues.
See Scheme 7.
3.4. Synthesis of l-threo C5-BODIPYTM-LacCer
analogue (33)
See Scheme 8.
4. Summary
The (2R,3R) (or d-threo, compound 8) and (2R,3S) (or
l-erythro, compound 20) sphingosines were synthesized
as outlined in Schemes 2 and 5, respectively, by the reac-
tion of (R)-Garner aldehyde (compound 4) (Garner et al.,
1988; Garner and Park, 1987; Garner and Park, 1992)
with lithium pentadecyne in the presence of zinc bromide
in Et2O or HMPA in THF (Herold, 1988), respectively.
(R)-Garner aldehyde was prepared (see Scheme 1) from
N-Boc-d-serine (1), which was converted to its methyl
ester and then treated with 2,2-dimethoxypropane in the
presence of p-toluenesulfonic acid in benzene, followed
by DIBAL-H reduction at −78 ◦C. The oxazolidine ring
was opened with Amberlyst 15 resin, and Red-Al reduc-
tion (Van Overmeire et al., 1999) of the propargylic
alcohol in Et2O afforded (2R,3R)- and (2R,3S)-N-Boc-
sphingosines (compounds 7 and 19, respectively). The
diazo transfer reaction afforded the stereoisomeric 2-
Acknowledgment
This work was supported in part by NIH grant HL-
083187.

Y. Liu, R. Bittman / Chemistry and Physics of Lipids 142 (2006) 58–69
69
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